Bone Regeneration Based on Tissue Engineering Conceptions — A 21st Century Perspective

The role of Bone Tissue Engineering in the field of Regenerative Medicine has been the topic of substantial research over the past two decades. Technological advances have improved orthopaedic implants and surgical techniques for bone reconstruction. However, improvements in surgical techniques to reconstruct bone have been limited by the paucity of autologous materials available and donor site morbidity. Recent advances in the development of biomaterials have provided attractive alternatives to bone grafting expanding the surgical options for restoring the form and function of injured bone. Specifically, novel bioactive (second generation) biomaterials have been developed that are characterised by controlled action and reaction to the host tissue environment, whilst exhibiting controlled chemical breakdown and resorption with an ultimate replacement by regenerating tissue. Future generations of biomaterials (third generation) are designed to be not only osteoconductive but also osteoinductive, i.e. to stimulate regeneration of host tissues by combining tissue engineering and in situ tissue regeneration methods with a focus on novel applications. These techniques will lead to novel possibilities for tissue regeneration and repair. At present, tissue engineered constructs that may find future use as bone grafts for complex skeletal defects, whether from post-traumatic, degenerative, neoplastic or congenital/developmental “origin” require osseous reconstruction to ensure structural and functional integrity. Engineering functional bone using combinations of cells, scaffolds and bioactive factors is a promising strategy and a particular feature for future development in the area of hybrid materials which are able to exhibit suitable biomimetic and mechanical properties. This review will discuss the state of the art in this field and what we can expect from future generations of bone regeneration concepts.     

Effect of excessive methionine on the development of the cranial growth plate in newborn rats

ObjectiveMethionine is an essential amino acid and pivotal for normal growth and development. However, previous animal studies have shown that excessive maternal intake of methionine causes growth restrictions, organ damages, and abnormal growth of the mandible in newborn animals. However, the effect of excessive methionine on the development of the cranial growth plate is unknown. This study investigated histological alterations of the cranial growth plate induced by high methionine administration in newborn rats.DesignTwenty pregnant dams were divided into a control and an experimental group. The controls received a diet for rats and the experimental group was fed from the 18th gestational day with a special manufactured high methionine diet for rats. The high methionine diet was maintained until the end of the lactation phase (day 20). The offspring of both groups were killed at day 10 or 20 postnatally and their spheno-occipital synchondroses were collected for histological analysis.ResultsThe weight of the high-dose methionine treated experimental group was considerably reduced in comparison to the control group at day 10 and 20 postnatally. The cartilaginous area of the growth plate and the height of the proliferative zone were markedly reduced at postnatal day 10 in the experimental group.ConclusionsIn summary, the diet-induced hypermethioninemia in rat dams resulted in growth retardations and histomorphological changes of the spheno-occipital synchondrosis, an important craniofacial growth centre in newborns. This finding may elucidate facial dysmorphoses reported in patients suffering from hypermethioninemia.     

Therapeutic alliance and behavior inhibition

Abstract

The study examined whether the quality of the therapeutic alliance is associated with a tendency toward behavior inhibition. Inhibition was measured by the frequency with which the clients used the word “not” and by their verbal productivity. In treatment sessions with low-quality therapeutic alliance, clients tended to use the word “not” more often and speak fewer words. Results suggest that it could be useful for therapists to use indexes of behavior inhibition as problem markers in the therapeutic alliance.     

A 2-year follow-up MRI study for the evaluation of an age estimation method based on knee bone development

Age estimation is an actual topic in the area of forensic medicine with a special focus on the age limits of 16 and 18 years. Current research on this topic relies on retrospective data of inhomogeneous populations relating to sex, age range, and socioeconomic status. In this work, we present a 2-year follow-up study for the evaluation of an age estimation method on a prospective magnetic resonance imaging (MRI) knee data collective of a homogeneous population. The study includes 40 male subjects from northern Germany aged 14 to 21 years. Three MRI examinations were evenly acquired within 2 years for each subject. As a first evaluation, a three-stage system was used to assess the ossification status of the knee (I:“open”, II:“partially ossified”, III:“fully ossified”). Three raters assessed the growth plate of the distal femur, proximal tibia, and proximal fibula based on central 2D slices. A good inter-rater agreement was attained (κ = 0.84). All subjects younger than 18 years were rated as stage I and had a cumulative knee score (SKJ) ≤ 5. Based on the follow-up datasets, new parameters quantifying the intra-individual ossification process were calculated. The results of this follow-up analysis show a different start, end, and speed of each growth plate’s maturation as well as an ossification peak for individuals at the age of 16. The generated MRI database provides new insights into the ossification process over time and serves as a basis for further evaluations of age estimation methods.

     

Assessing the sensitivity of diffusion MRI to detect neuronal activity directly

Functional MRI (fMRI) is widely used to study brain function in the neurosciences. Unfortunately, conventional fMRI only indirectly assesses neuronal activity via hemodynamic coupling. Diffusion fMRI was proposed as a more direct and accurate fMRI method to detect neuronal activity, yet confirmative findings have proven difficult to obtain. Given that the underlying relation between tissue water diffusion changes and neuronal activity remains unclear, the rationale for using diffusion MRI to monitor neuronal activity has yet to be clearly established. Here, we studied the correlation between water diffusion and neuronal activity in vitro by simultaneous calcium fluorescence imaging and diffusion MR acquisition. We used organotypic cortical cultures from rat brains as a biological model system, in which spontaneous neuronal activity robustly emerges free of hemodynamic and other artifacts. Simultaneous fluorescent calcium images of neuronal activity are then directly correlated with diffusion MR signals now free of confounds typically encountered in vivo. Although a simultaneous increase of diffusion-weighted MR signals was observed together with the prolonged depolarization of neurons induced by pharmacological manipulations (in which cell swelling was demonstrated to play an important role), no evidence was found that diffusion MR signals directly correlate with normal spontaneous neuronal activity. These results suggest that, whereas current diffusion MR methods could monitor pathological conditions such as hyperexcitability, e.g., those seen in epilepsy, they do not appear to be sensitive or specific enough to detect or follow normal neuronal activity.Developing a direct MRI method to detect neuronal activity in vivo and noninvasively is a major focus in neuroscience. Progress in this area is required to improve our understanding of normal brain function, and in a clinical setting, to develop new tools for studying normal and abnormal development to diagnose diseases and disorders of the brain. Functional MRI (fMRI) has been widely used in the cognitive neurosciences since its invention in the 1990s (1–3). The most widely used fMRI method, blood-oxygenation-level-dependent (BOLD) MRI, detects hemodynamic changes in the brain, which only indirectly reflects neuronal activity. Moreover, its hemodynamic origin limits both its spatial and temporal resolution and its interpretation as a direct proxy for neuronal activity (4, 5).More recently, several MRI methods were proposed to provide more direct measures of neuronal excitation (6). In particular, diffusion MRI, a method to measure the apparent diffusivity of water within tissues (7–9), has been suggested as a direct functional imaging method to detect neuronal activity (10–13). Early in vivo experiments in both humans and animals reported small but significant increases in highly diffusion-weighted MRI signals, which were ascribed to changes directly induced by the underlying neuronal activity rather than indirect hemodynamic changes (10–13). In vitro experiments on brain slices (14, 15) and spinal cord (16) reported similar reductions in water diffusivity under conditions of extreme hyperexcitability using strong pharmacologic stimulants.However, functional diffusion MRI (fDMRI) has not been widely used or adopted since its introduction almost two decades ago. Two major reasons for this may be a dearth of experiments that convincingly establish its neurophysiological basis and the poor reproducibility of the originally reported changes in diffusion MRI signals by different laboratories. The inability to detect the predicted changes using fDMRI and the possible confounds of hemodynamic contributions in fDMRI measurements in vivo do not argue for a robust connection between changes in diffusion MRI and underlying neuronal activity (17–20). Thus, “ground-truth” experiments, potentially establishing a connection between the changes in diffusion MRI and underlying neuronal activity, are needed, particularly to shed light on the possible biophysical basis of the fDMRI signal.Recently, we developed a novel test bed that could be used to assess non–hemodynamic-based functional MRI methods, in which MR signal acquisition and intracellular calcium fluorescence imaging to monitor neuronal activity can be performed simultaneously on organotypic cortical cultures from rat brains (21). The organotypic cortex culture is a well-characterized biological model of neuronal activity free of hemodynamic, respiratory, and other physiological confounds. Not only is the in vivo cortical cytoarchitecture preserved (including cortical layers and cortical cell types), but neuronal activity in the culture also displays bursts of spontaneous neuronal avalanches grouped into so-called up-states and separated by periods of low activity (22–25), resembling resting neuronal activity in vivo (26–28). Specifically, fluorometric calcium (Ca²⁺) imaging is used to detect intracellular Ca²⁺ concentration changes that closely follow action potentials in neurons under normal conditions and provide a direct method for detecting neuronal spiking activity in a neuronal network (29, 30). This test bed thus allows one to study precisely and accurately temporal correlations between the candidate functional MR signals, which are free of the usual in vivo confounds, and the underlying neuronal spiking activity by using an independent intracellular Ca²⁺ imaging experiment (21).In the current study, diffusion MR signals are obtained simultaneously with intracellular calcium fluorescence imaging of the organotypic cortex culture. The direct effects of neuronal activity on the diffusion MR signals are studied by time-series analysis of the simultaneous calcium and MR signals during normal neuronal activity and in different pathological states, which include induced hyperexcitability by kainic acid (kainate) and potassium, disinhibition by picrotoxin (PTX), suppression of excitability by tetrodotoxin (TTX), and cell volume modulation caused by osmotic pressure challenges. On the basis of these findings, it is possible to assess the prospect of detecting normal and abnormal neuronal activity using fDMRI and to better understand the relationship between fDMRI changes and biophysical mechanisms associated with neuronal excitation.     

Management of diabetes in pregnancy: comparison of guidelines with current practice at Austrian and Australian obstetric center

Aim

To compare Austrian and Australian national guidelines for gestational and pre-gestational diabetes and estimate the level to which physicians comply with their country’s guidelines.

Methods

Austrian (ÖDG, Austrian Diabetes Society) and Australian guidelines (ADIPS, Australasian Diabetes in Pregnancy Society) for the treatment of gestational diabetes and pre-gestational diabetes were systematically reviewed. Current practices in two obstetric centers in Austria and Australia were assessed by interviewing key stakeholders through questionnaires assessing different components of diabetes care. For gestational diabetes, these components were screening, abnormal oral glucose tolerance test values (mmol/L), abnormal values for diagnosis, further management when abnormal values are detected, monitoring/glucose targets (mmol/L), further management and indications for insulin therapy, route and timing of delivery, and postpartum management and counseling. For pre-gestational diabetes, the components were management during the preconceptional period, glucose target values, medical surveillance, obstetric surveillance, medication used, route and timing of delivery, and postpartum management and counseling.

Results

More variation was found in the management of gestational than pre-gestational diabetes. There were differences in oral glucose tolerance test and cut-off levels for diagnosing gestational diabetes in both centers and guidelines. Australian guidelines recommended two-stage screening for gestational diabetes, while Austrian guidelines recommended one-stage screening. At the Austrian obstetric center, amniocentesis was recommended for determining the start of insulin treatment in pregnant women with gestational diabetes. This approach was neither used at the Australian obstetric center nor recommended by any of the two guidelines.

Conclusion

Our study showed that it was difficult to standardize screening criteria and diagnostic methods for gestational and pre-gestational diabetes. National and international consensus has yet to be achieved in the management of diabetes in pregnancy.The number of cases of diabetes worldwide has significantly increased in the last decade and it is expected to double by 2030 (1). This “diabetic epidemic” also considerably affects pregnant women (2). However, the management of pre-gestational and gestational diabetes, the latter being defined as glucose intolerance first detected in pregnancy, remains controversial (3). Gestational and pre-gestational diabetes are associated with increased feto-maternal morbidity, including stillbirth, macrosomia, and fetal malformations, as well as long-term complications in the mother and offspring (4-6). However, treatment and/or monitoring reduce perinatal mortality to the rate in the healthy population. There is no internationally agreed approach and there are neither up-to-date World Health Organization (WHO) recommendations nor fact sheets designed especially for diabetes in pregnancy. The complexity of gestational and pre-gestational diabetes, its underlying pathogenetic mechanism, and recent insights into potential and far-ranging complications have justified the establishment of a considerable number of recent national guidelines (7). Variation in treatment strategies has originated from different views, approaches, and traditional management in obstetric clinics around the globe.As a novelty, this study does not only compare national guidelines of Austria and Australia, two developed high-income countries situated on different continents, but also estimates the level to which physicians comply with their country’s guidelines. Since currently no international standardized approach to screening criteria and diagnostic methods for gestational diabetes and pre-gestational diabetes exists and opinions differ even on the national level, we hypothesized that there were major differences in screening, diagnosing, and treating diabetes in pregnancy. An additional aim of this study was to produce a table of requirements that should be incorporated into future guidelines.     

Occurrence and co-localization of amyloid beta-protein and apolipoprotein E in perivascular drainage channels of wild-type and APP-transgenic mice

The deposition of the amyloid beta-protein (Abeta) is a hallmark of Alzheimer's disease (AD). One reason for Abeta-accumulation and deposition in the brain may be an altered drainage along perivascular channels. Extracellular fluid is drained from the brain towards the cervical lymph nodes via perivascular channels. The perivascular space around cerebral arteries is the morphological correlative of these drainage channels. Here, we show that Abeta is immunohistochemically detectable within the perivascular space of 25 months old wild-type and amyloid precursor protein (APP)-transgenic mice harboring the Swedish double mutation driven by a neuron specific promoter. Only small amounts of Abeta can be detected immunohistochemically in the perivascular space of wild-type mice. Cerebrovascular and parenchymal Abeta-deposits were absent. In APP-transgenic mice, large amounts of Abeta were found in the perivascular drainage channels accompanied with cerebrovascular and parenchymal Abeta-deposition. The apolipoprotein E (apoE) immunostaining within the perivascular channels did not vary between wild-type and APP-transgenic mice. Almost 100% of the area that represents the perivascular space was stained with an antibody directed against apoE. Here, Abeta co-localized with apoE indicating an involvement of apoE in the perivascular clearance of Abeta. Fibrillar congophilic amyloid was not seen in wild-type mice. In APP-transgenic animals, congophilic fibrillar amyloid material was seen in the wall of cerebral blood vessels but not in the perivascular space. In conclusion, our results suggest that non-fibrillar forms of Abeta are drained along perivascular channels and that apoE is presumably involved in this clearance mechanism. Overloading such a clearance mechanism in APP-transgenic mice appears to result in insufficient Abeta-clearance, increased Abeta-levels in the brain and the perivascular drainage channels, and finally in Abeta-deposition. In so doing, our results strengthen the hypothesis that an alteration of perivascular drainage supports Abeta-deposition and the development of AD.     

A comprehensive in vitro investigation of a portable magnetic separator device for human blood detoxification

A portable magnetic separator device is being developed for a proposed magnetically based detoxification system. In this paper, the performance of this device was evaluated via preliminary in vitro flow experiments using simple fluids and a separator unit consisting of one tube and two metal wires, each at the top and bottom of the tube. The effects of the following factors were observed: mean flow velocity U(o) (0.14-45 cm s(-1)), magnetic field strength micro(o)H(o) (0.125-0.50 T), wire size R(w) (0.125, 0.250 and 0.500 mm), wire length L(w) (2, 5 and 10 cm), wire materials (nickel, stainless steel 304 and 430) and tube size (outer radius R(o) = 0.30 mm and inner radius R(i) = 0.25 mm; R(o) = 0.50 mm and R(i) = 0.375 mm; and R(o) = 2.0 mm and R(i) = 1.0 mm). Our observations showed that the experimental results fit well with the corresponding theoretical results from the model we previously developed at a low flow velocity area (for example, U(o) < or = 20 cm s(-1)), strong external magnetic field (for example, > or = 0.30 T) and long wire length (for example, L(w) = 10 cm). The experimental results also showed that more than 90% capture efficiency is indeed achievable under moderate systemic and operational conditions. Pressure drop measurements revealed that the device could work well under human physiological and clinical conditions, and sphere buildup would not have any considerable effect on the pressure drop of the device. The breakthrough experiments demonstrated that a lower flow rate V, higher applied magnetic field micro(o)H(o) and diluted sphere suspension, i.e. lower C(o), would delay the breakthrough. All the results indicate the promise of this portable magnetic separator device to efficiently in vivo sequestrate nano-/micro-spheres from blood flow in the future magnetically based detoxification system.